A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina
ISSN: 2955 – 1145 (print); 2955 – 1153 (online)
ORIGINAL RESEARCH ARTICLE
*Hauwa Salisu Usman1, Shehu Muhammad Hassan 1, Idris Zubairu Sadiq1 , Christiana Oyetola Olawoyin1 , Murja Ibrahim Danja2 and Abdullahi Balarabe Sallau1
1Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria
2 Federal University of Education, Zaria, Nigeria
*Corresponding author: Hauwa Salisu Usman 
[email protected]
Advanced glycation end-products (AGEs) are the final products derived from the Maillard reaction, a non-enzymatic glycation of free amino groups by sugars and aldehydes. AGE formation begins under hyperglycemic or oxidative stress conditions. This work aimed to determine the anti-glycation and antioxidant potential of leaves and fruit pulp of Dialium guineense (black velvet tamarind). Plant materials were extracted with chloroform, methanol, and ethyl acetate using the maceration technique. Qualitative phytochemical screening was carried out alongside in vitro antioxidant assay (DPPH radical scavenging) and anti-glycation assay via the BSA-glucose model using spectrofluorescence, with aminoguanidine as the positive control. All experiments were carried out in triplicate, with data presented as mean ± standard deviation (SD), and analyzed using one-way analysis of variance (ANOVA) in SPSS version 20. Phytochemicals identified include saponins, cardiac glycosides, steroids, triterpenes, flavonoids and tannins. Antioxidant activity showed that the DG methanol leaf extract had the highest activity (72.44% at 250 µg/mL), which was highly significant (p < 0.05) compared to the control (92.01%). For the fruit pulp, the chloroform fruit extract had the highest activity (67.09% at 500 µg/mL). Anti-glycation activity of DG leaves showed that the methanol leaf extract had the highest activity (86.46% at 125µg/mL); for the fruit pulp, ethylacetate fruit extract had the highest activity (95.62% at 250µg/mL), which was highly significant (p < 0.05) compared to the control (83.68%). Despite the remarkable outcome achieved, some limitations of this pilot study include the exclusion of quantitative phytochemical analysis and other assay variants, and the scope being confined to an in vitro medium. In conclusion, the dual potential of both fruits and leaves of Dialium guineense in anti-glycation and antioxidant studies adds to its pharmacological value as a potential functional food or medicinal plant.
Keywords: Antiglycation, Antioxidant, Phytochemicals and Dialium guineense
For the first time, the antiglycation activity of Dialium guineense extract was reported
Anti-glycation activity was conducted using the BSA-glucose in vitro model through spectrofluorescence
Phytoconstituent evaluation showcased numerous therapeutic bioactives
Plant extracts revealed remarkably significant antioxidant and antiglycation activity
The rising incidence of lifestyle-related illnesses has spurred numerous studies on dietary antioxidants. Excessive production of chemicals resulting from the non-enzymatic multi-stage glycation process is a consequence of sedentary lifestyles, hypercaloric and high-fructose diets, and the increasing consumption of processed food components. Furthermore, advanced glycation end products (AGEs) have been shown to have a significant role in the pathogenesis of a number of metabolic disorders linked to chronic illnesses, including diabetes, cardiovascular disease, atherosclerosis, and its repercussions (Nowotny et al., 2018; Zawada et al., 2022).
The accumulation of Advanced Glycation End Products (AGEs) has been a significant focus of research on metabolic disorders. Glycation, a non-enzymatic process in which reducing sugars react with amino groups of proteins, lipids, and nucleic acids, results in the formation of complex molecules known as AGEs. Cardiovascular disease, dementia, and renal dysfunction are among the chronic illnesses associated with their prevalence (Twarda-Clapa et al., 2022). AGEs' pathophysiological effects are mediated by receptor interactions, especially with the Receptor for Advanced Glycation End Products (RAGE), which intensifies oxidative stress and triggers inflammatory responses (Chinchansure et al., 2015; Sadeghi et al., 2023).
Our body produces free radicals, such as reactive oxygen and nitrogen species, through a variety of endogenous systems, exposure to various physicochemical conditions, or pathological conditions. For healthy physiological function, free radicals and antioxidants must be in equilibrium. Oxidative stress results when the body's capacity to control free radicals is exceeded. As a result, free radicals damage proteins, lipids, and DNA, leading to a variety of illnesses in humans. Therefore, applying antioxidants from outside sources can help manage this oxidative damage. By neutralizing these free radicals, antioxidants shield cells from oxidative damage (Lobo et al., 2010). The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging test is commonly recognized as a dependable and simple technique to evaluate the antioxidant potential of different compounds (Abuh et al., 2022; Inuwa et al., 2024; Isah et al., 2025; Komolafe et al., 2024; Muhammad, I. et al., 2024; H. S. Muhammad et al., 2024; I. Muhammad et al., 2025; Muhammad I. et al., 2024; I. Muhammad & Lawal, 2025; Musa & Imam, 2022; Nasir et al., 2025; Ndu et al., 2025). It enables a preliminary evaluation of the extracts' potential health benefits by measuring their capacity to donate hydrogen atoms to neutralize free radicals (Brand-Williams et al., 1995).
Due to their ability to reduce oxidative stress and stop or limit the development of AGE accumulation, natural compounds have become more popular in the search for potent anti-glycation therapies (Elosta et al., 2012). Dialium guineense (DG), also known as Black Velvet Tamarind, has emerged as one of the many botanical resources that merit further investigation. Native to West Africa, this fruit-bearing tree has long been used in ethnomedicine, especially for its reported anti-inflammatory, anti-oxidative, and anti-diabetic qualities (Lokonon et al., 2013; Besong et al., 2016; Oluwole-Banjo, 2019).
Plate 1: Dialium guineense fruit, fruit pulp, seeds and leaves.
Known as "Awin" in Yoruba, "Icheku" in Igbo, and "Tsamiyar kurmi" in Hausa, Dialium guineense (DG), or black velvet tamarind, is found in a number of West African nations, including Ghana, Sierra Leone, Senegal, and Nigeria (Besong et al., 2016). The fruit is sold in local markets and is frequently eaten as a snack. Occasionally, it is used to make a non-alcoholic beverage. Furthermore, the gum from the seeds is used in Japanese cooking to improve food viscosity, and the blossoms and leaves are eaten as vegetables (Utubaku et al., 2017). The tree's leaves alleviate fever and oedema, while its bark is used to treat conditions such as cancer, headaches, and dental hygiene (Ovuakporie-Uvo et al., 2015). According to Besong et al. (2016), the fruits are traditionally used to cure wounds, malaria, pneumonia, diarrhoea, and cough. It has also been reported to help breastfeeding women in southeast Nigeria prevent genital infections. The plant is known for its saponin content, which may help promote dental health by reducing plaque and cavities (Alagbe et al., 2020).
To the best of our knowledge, this is the first research on the antiglycation activity of Dialium guineense fruit and leaf extracts. Despite a few existing studies on the antioxidant activity of the plant, all were based on methanol or aqueous extracts (Adopo et al., 2024; Ebo et al., 2025; Ogu et al., 2013) or an n-hexane/ethanol extract (Dressman et al., 2024), without considering various solvent systems. Moreover, the review on the medicinal importance of DG (Besong et al., 2016) lacks reports on its antiglycation activity; hence, the need to bridge this research gap. This study aims to evaluate the anti-glycation and antioxidant properties of DG fruit and leaf extracts in vitro using different solvent systems, to assess the specific activity of the various solvents based on their polarity. Assessing their efficacy in inhibiting AGE formation and scavenging DPPH radicals will elucidate the therapeutic potential of this indigenous resource.
Methanol, Ethyl-acetate, Chloroform, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Phosphate buffer, Ascorbic acid, Bovine serum albumin (BSA), Glucose, Phosphate-buffered saline (PBS), Trichloroacetic acid, Aminoguanidine, Distilled water, Test tubes, Beaker, Measuring cylinder, Pipette, Glass rod
Dialium guineense leaves and fruits were collected from a local market in Sabon gari, Zaria, Kaduna State. Plant samples were authenticated at the Herbarium Unit of the Department of Botany, Faculty of Life Science, Ahmadu Bello University, Zaria, Kaduna State, Nigeria, where a voucher number (ABU0243) was deposited.
Air-dried whole plant materials were ground into a fine powder using a mortar and pestle. Powdered plant material (10 g) was extracted separately using 150ml each of methanol, ethyl-acetate, and chloroform. The supernatants were filtered using a Whatman No. 1 filter paper into pre-weighed glass vials and air-dried. The quantity of plant materials extracted was determined and stored in air-tight glass vials in the dark until usage.
The presence/absence of phytochemicals in DG fruit and leaf extracts was determined using colour change or precipitate formation assays, including flavonoids (Shinoda Method), phenolic compounds (Ferric chloride), alkaloids (Dragendorff, Wagner, and Mayer Methods), triterpenes and steroids (Liebermann–Buchard Method). The results were expressed as the relative presence of the phytochemicals, considering the following symbols: presence [+] and absence [-] (Harborne, 1998; Evans, 2002).
The antioxidant power of plant extracts was determined using the DPPH free radical scavenging assay as described by Shah et al. (2013). Briefly, 0.1 ml each of methanol, 1 mg/ml ascorbic acid, and 1 mg/ml plant extract was added, in triplicate, to the control, standard, and extracts (methanol, ethyl acetate, and chloroform), respectively. Thereafter, 3 mL of 0.24 mg/mL DPPH (prepared in methanol) was added to the test tubes. The mixture was then incubated for 5 min, followed by 30 min in the dark at 25 °C. Five different concentrations (62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL) of the control and plant samples were prepared in triplicate. The absorbance was read at 517 nm using an Agilent Cary 60 UV-Vis Spectrophotometer. Distilled water was used as the blank control to reset the instrument prior to each absorbance measurement. The percentage antioxidant or free radical scavenging activity of the extracts and ascorbic acid was determined by substituting absorbance values using the formula below:
\(Antioxidant\ activity\ (\%\ ) = \ \frac{Absorbance\ of\ control - Absorbance\ of\ test}{Absorbance\ of\ control} \times 100\)
The antiglycation activity of the extracts was estimated using the methods of Matsuura et al. (2002) and Kaewnarin et al. (2014). In brief, 20 µl each of 800 µg/ml BSA and 200 mM D-glucose were added, in triplicate, to test tubes labelled 'standard' and 'plant extracts' (methanol, ethyl acetate, and chloroform). Thereafter, 20 µl each of 50 mM phosphate buffer (pH 7.4) containing 0.2 g/l sodium azide was added to the test tubes labelled standard and the various plant extracts mentioned above. 1 mg/ml of both aminoguanidine and plant extract (prepared in phosphate buffer containing sodium azide) was added to test tubes labelled 'standard' and 'plant extracts,' respectively. Five different concentrations (62.5 µg/mL, 125 µg/mL, 250 µg/mL, 500 µg/mL, and 1000 µg/mL) of the control and plant samples were prepared in triplicate. Afterwards, the mixture was incubated at 37°C for 7 days. The fluorescence intensity was measured at an excitation wavelength of 370 nm and an emission wavelength of 440 nm using an Agilent Cary Eclipse Fluorescence Spectrometer. Distilled water was used as the blank control to reset the instrument prior to each absorbance measurement. The percentage antiglycation activity of the extracts and the control was calculated using mean absorbance values from the plant extracts and the control substituted in the formula below:
\(Antiglycation\ activity\ (\%\ ) = \ \frac{Fluorescence\ intensity\ of\ control - Fluorescence\ intensity\ of\ test}{Fluorescence\ intensity\ of\ control} \times 100\)
All experiments were carried out in triplicate, with data presented as mean ± standard deviation (SD), and analyzed using one-way analysis of variance (ANOVA) in Statistical Package for the Social Sciences (SPSS) version 20 for Windows. A Duncan post hoc test was conducted to detect differences in the means of the various test solutions. P value less than 0.05 (p < 0.05) was considered statistically significant.
Phytochemicals identified in ethyl acetate, chloroform, and methanol extracts of DG leaves and fruits included saponins, triterpenes, tannins, and flavonoids. Moreover, steroids and triterpenes were present in all the plant extracts evaluated. However, anthraquinones and alkaloids were not detected in all the plant extracts (Table 1).
Table 1: Phytochemical constituents of Dialium guineense leaves and fruits.
| Phytochemicals | DGMLE | DGELE | DGCLE | DGMFE | DGEFE | DGCFE |
|---|---|---|---|---|---|---|
| Saponins | + | + | - | + | - | - |
| Cardiac glycosides | + | + | - | + | + | - |
| Steroids and triterpenes | + | + | + | + | + | + |
| Flavonoids | - | - | + | + | - | - |
| Tannins | + | - | + | - | - | + |
| Anthraquinones | - | - | - | - | - | - |
| Alkaloids | - | - | - | - | - | - |
KEY: DGMLE : Dialium guineense methanol leaf extract DGELE : Dialium guineense ethylacetate leaf extract DGCLE : Dialium guineense chloroform leaf extract DGMFE : Dialium guineense methanol fruit extract DGEFE : Dialium guineense ethylacetate fruit extract DGCFE : Dialium guineense chloroform fruit extract - : showing the absence of a phytochemical + : showing the presence of a phytochemical
The DPPH scavenging activity of DG fruit extract is depicted in Figure 1. The chloroform extract had the highest activity (67.09% at 500 µg/mL), which was significantly lower (p < 0.05) than that of ascorbic acid (control). Comparatively, leaf methanol extract displayed a higher antioxidant activity (72.44% at 250µg/mL) compared to ethylacetate (57.89%) and chloroform extract (50.85%) (Figure 2). However, DPPH radical scavenging capacity between the methanol leaf extract and ascorbic acid was significantly low (p < 0.05). All data were presented as mean ± SD of triplicate values. a-c values with different alphabets over the bars are significantly (p < 0.05) different from each other.
Figure 1: In vitro DPPH Radical Scavenging Activity of Dialium guineense Fruit Extracts: data are shown as percentage DPPH scavenging activity. Data are presented as mean ± SD of triplicate values. a-c values with different alphabets over the bars are significantly (p < 0.05) different from each other.
Figure 2: In vitro DPPH Radical Scavenging Activity of Dialium guineense Leaf Extracts: data are shown as percentage DPPH scavenging activity. Data are presented as mean ± SD of triplicate values. a-d values with different alphabets over the bars are significantly (p < 0.05) different from each other.
Results from the antiglycation activity of Dialium guineense fruits is depicted in Figure 3. DG ethyl acetate fruit extract had the highest antiglycation activity (95.20% at 250 µg/mL) compared to chloroform (91.78%) and methanol (55.19%); however, this was significantly higher (p < 0.05) than the control (aminoguanidine). Methanol leaf extract had the most antiglycation activity (86.48% at 125µg/mL) as shown in (Figure 4); however, this was significantly (p < 0.05) high compared to the control (aminoguanidine). All data were presented as mean ± SD of triplicate values. a-d values with different alphabets over the bars are significantly (p < 0.05) different from each other.
Figure 3: In vitro Antiglycation Activity of Dialium guineense Fruit Extracts: data are shown as percentage antiglycation activity. Data are presented as mean ± SD of triplicate values. a-c values with different alphabets over the bars are significantly (p < 0.05) different from each other.
Figure 4: In vitro Antiglycation Activity of Dialium guineense Leaf Extracts: data are shown as percentage antiglycation activity. Data are presented as mean ± SD of triplicate values. a-d values with different alphabets over the bars are significantly (p < 0.05) different from each other.
The present study evaluated the potential of Dialium guineense (DG) fruit and leaf extracts to inhibit advanced glycation end products (AGEs) and to scavenge DPPH radicals in vitro. The results suggest that both fruit and leaf extracts possess notable anti-glycation and antioxidant properties, which may offer therapeutic value in the management of conditions associated with oxidative stress, such as diabetes and ageing.
To the best of our knowledge, there is no existing literature on the antiglycation activity of DG fruits and leaves; hence, the need for this pilot study. In our findings, both the fruit and leaf extracts demonstrated remarkable antiglycation activity, consistent with previous studies on other plant-based extracts (Bi et al., 2011; Silva et al., 2022; Usman et al., 2023b, 2025a). However, the fruit extract showed higher efficacy (95.20% at 250 µg/mL) than the control; this may be attributed to the different solvent system used. Chloroform and ethyl acetate fruit extracts had very high activity values (95% and 91%, respectively), suggesting that the bioactive principles present in these extracts are non-polar/moderately polar in nature. The leaf extract also exhibited remarkable antiglycation activity (86%). Similarly, extracts from numerous plant species, such as Ficus racemosa, Ficus botryocarpa and Solanum macrocarpon have shown promising anti-glycation effects (Cushnie et al., 2024; Usman et al., 2025b). The potential of DG extracts in this regard could offer an alternative or adjunctive approach to managing AGE-related complications, since AGEs are implicated in the pathogenesis of several chronic diseases, including diabetes, cardiovascular disease, and Alzheimer's disease (Khalid et al., 2022).
The DPPH free radical scavenging activity is a widely used model for evaluating the free radical scavenging ability of various compounds. Absorbance decreased at 517 nm, resulting in a colour change from purple to yellow, as radicals were scavenged by antioxidants through the donation of hydrogen to form the stable DPPH molecule (Nandhakumar and Indumathi, 2013). In our study, the leaf methanol extract of DG exhibited superior scavenging activity on DPPH radical when compared to the fruit extract. This can be attributed to the presence of phytochemicals, such as saponins, cardiac glycosides, tannins, steroids, and triterpenes, in the leaves. Saponins, in particular, are known for their electron-donating properties, which enable them to neutralize free radicals efficiently. Saponins, from a variety of sources, have also been shown to have hypocholesterolemic, anticoagulant, anticarcinogenic, hepatoprotective, hypoglycemic, immunomodulatory, neuroprotective, anti-inflammatory, and antioxidant activities (Rao and Gurfinkel, 2000). Our findings align with those reported by other researchers, who have observed significant DPPH scavenging activity in various plant extracts, including those from Moringa peregrina, Crataegus meyeri, Vernonia amygdalina, Carissa edulis, Diospyros mespiliformis, and Centella asiatica (Dehshahri et al., 2012; Ekin et al., 2017; Hussen & Endalew, 2023; Usman et al., 2023a; Kalemba et al., 2024). Interestingly, the DPPH scavenging activity of the fruit extract, although slightly lower than that of the leaf extract, still demonstrated promising results. This suggests that DG leaves could be an alternative source of antioxidants, particularly in regions where fruit availability may be limited.
However, some key constraints of our in vitro-based studies include a lack of systemic interactions, poor metabolic simulation, inability to model chronic/long-term effects, and high variability between laboratories (Ghallab, 2013; Tice et al., 2013).
While this pilot study provides valuable insights into the bioactivity of DG fruit and leaf extracts, further research is necessary to explore the molecular mechanisms underlying their anti-glycation and antioxidant activities. It would be beneficial to identify and isolate the specific bioactive compounds responsible for these effects. Additionally, in vivo studies are needed to confirm DG's therapeutic potential in animal models, followed by clinical trials to assess its efficacy and safety in humans. Furthermore, future studies should examine compound isolation and identification through LC-MS and orthogonal AGE assays. The emerging field of polyherbal medicine could offer enhanced therapeutic strategies by leveraging the complementary bioactive profiles of different plants.
Our preliminary findings indicate dual potential for both the fruit and leaf extracts for anti-glycation and antioxidant activities. These findings add to the pharmacological value of DG as a functional food or medicinal plant, offering a potential protective effect against oxidative stress-related diseases, including cancer, cardiovascular diseases, and neurodegeneration. By establishing a scientific basis for DG's traditional medicinal uses, this study emphasizes the need for further research into the implications of Dialium guineense in preventing oxidative stress-related complications.
We highly appreciate the immense assistance of Malam Aliyu Mansur of Mary Hallaway Teaching Laboratories, Department of Biochemistry, ABU, Zaria. We are grateful to the Academic technologists in Ahmadu Bello University's multi-user laboratory for their help and support. We also acknowledge Malam Kabiru in the Department of Pharmacognosy and Drug Development's research laboratory at Ahmadu Bello University for his assistance and support.
The authors declare no conflict of interest.
Conceptualisation: HSU, ABS; Laboratory experiments: COO, HSU; Data Analysis: COO, HSU, SMH, ISZ; Writing- original draft preparation: HSU, COO; Writing-review and editing: HSU, ISZ, SMH, MID, ABS; Resources: HSU, COO, SMH, MID, ABS; Supervision: HSU, ABS. All authors approved the final version of the manuscript.
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